Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket
Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium...
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2023
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| Online Access: | http://dx.doi.org/10.1016/j.bbagen.2023.130313 https://www.sciencedirect.com/science/article/pii/S0304416523000119 http://hdl.handle.net/10576/39329 |
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| _version_ | 1857415085872381952 |
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| author | Angelos, Thanassoulas |
| author2 | Vassilakopoulou, Vyronia Calver, Brian L. Buntwal, Luke Smith, Adrian Lai, Christopher Kontogianni, Iris Livaniou, Evangelia Nounesis, George Lai, F. Anthony Nomikos, Michail |
| author2_role | author author author author author author author author author author |
| author_facet | Angelos, Thanassoulas Vassilakopoulou, Vyronia Calver, Brian L. Buntwal, Luke Smith, Adrian Lai, Christopher Kontogianni, Iris Livaniou, Evangelia Nounesis, George Lai, F. Anthony Nomikos, Michail |
| author_role | author |
| dc.creator.none.fl_str_mv | Angelos, Thanassoulas Vassilakopoulou, Vyronia Calver, Brian L. Buntwal, Luke Smith, Adrian Lai, Christopher Kontogianni, Iris Livaniou, Evangelia Nounesis, George Lai, F. Anthony Nomikos, Michail |
| dc.date.none.fl_str_mv | 2023-01-31T10:52:39Z 2023-04-30 |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://dx.doi.org/10.1016/j.bbagen.2023.130313 Thanassoulas A, Vassilakopoulou V, Calver BL, et al. Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket [published online ahead of print, 2023 Jan 21]. Biochim Biophys Acta Gen Subj. 2023;1867(4):130313. doi:10.1016/j.bbagen.2023.130313 03044165 https://www.sciencedirect.com/science/article/pii/S0304416523000119 http://hdl.handle.net/10576/39329 4 1867 |
| dc.language.none.fl_str_mv | en |
| dc.publisher.none.fl_str_mv | Elsevier |
| dc.rights.none.fl_str_mv | http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Calmodulin Ryanodine receptor RyR2 Arrhythmias Cardiac disease |
| dc.title.none.fl_str_mv | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| dc.type.none.fl_str_mv | Article info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/article |
| description | Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2+) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaMN98I, CaMD132E, CaMD134H and CaMQ136P) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca2+, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | qu_8b75ce4ef6521c4b5613c5cec2653bcb |
| identifier_str_mv | Thanassoulas A, Vassilakopoulou V, Calver BL, et al. Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket [published online ahead of print, 2023 Jan 21]. Biochim Biophys Acta Gen Subj. 2023;1867(4):130313. doi:10.1016/j.bbagen.2023.130313 03044165 4 1867 |
| language_invalid_str_mv | en |
| network_acronym_str | qu |
| network_name_str | Qatar University repository |
| oai_identifier_str | oai:qspace.qu.edu.qa:10576/39329 |
| publishDate | 2023 |
| publisher.none.fl_str_mv | Elsevier |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | http://creativecommons.org/licenses/by/4.0/ |
| spelling | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocketAngelos, ThanassoulasVassilakopoulou, VyroniaCalver, Brian L.Buntwal, LukeSmith, AdrianLai, ChristopherKontogianni, IrisLivaniou, EvangeliaNounesis, GeorgeLai, F. AnthonyNomikos, MichailCalmodulinRyanodine receptorRyR2ArrhythmiasCardiac diseaseCalmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2+) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaMN98I, CaMD132E, CaMD134H and CaMQ136P) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca2+, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.We are grateful to Xuexun Fang (Laboratory of Molecular Enzymology and Enzyme Engineering of the Ministry of Education, Jilin University, China) for providing the pHSIE vector.Elsevier2023-01-31T10:52:39Z2023-04-30Articleinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://dx.doi.org/10.1016/j.bbagen.2023.130313Thanassoulas A, Vassilakopoulou V, Calver BL, et al. Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket [published online ahead of print, 2023 Jan 21]. Biochim Biophys Acta Gen Subj. 2023;1867(4):130313. doi:10.1016/j.bbagen.2023.13031303044165https://www.sciencedirect.com/science/article/pii/S0304416523000119http://hdl.handle.net/10576/3932941867enhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:qspace.qu.edu.qa:10576/393292024-07-23T13:52:21Z |
| spellingShingle | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket Angelos, Thanassoulas Calmodulin Ryanodine receptor RyR2 Arrhythmias Cardiac disease |
| status_str | publishedVersion |
| title | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_full | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_fullStr | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_full_unstemmed | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_short | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_sort | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| topic | Calmodulin Ryanodine receptor RyR2 Arrhythmias Cardiac disease |
| url | http://dx.doi.org/10.1016/j.bbagen.2023.130313 https://www.sciencedirect.com/science/article/pii/S0304416523000119 http://hdl.handle.net/10576/39329 |